Acetylcholine (ACh) is a major neurotransmitter in the central and peripheral nervous system. The rate-limiting step in ACh synthesis is believed to be the presynaptic acquisition of the precursor choline, achieved by the high-affinity, Na+-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). Recent breakthroughs in the molecular elucidation of invertebrate and mammalian CHT genes, the development of CHT-specific antibodies, and the creation of tractable in vitro and transgenic model systems have established new opportunities to define neuronal CHT subcellular distribution, mechanisms of activity- and receptor-dependent CHT regulation, and the functional consequences of genetic manipulation of CHT. Recently, we have established that CHT is predominantly vesicular in localization, both in vivo as well as in in vitro model systems. CHT appears to reside in a subpopulation of cholinergic synaptic vesicles that express the vesicular ACh transporter (VAChT) and which store ACh. Preliminary studies document both a change in the localization of CHT in synaptic membranes in response to depolarization in wildtype mice and a posttranslational mechanism to achieve normal levels of choline transport and HC-3 binding despite a 50% reduction in CHT protein in CHT +/- mice. In this new application, we apply biochemical, imaging and functional methodologies using in vitro and in vivo model systems to investigate the nature of the vesicular pool harboring CHT and clarify the physical requirements for vesicular targeting and synaptic CHT trafficking. Secondly, we explore plasma membrane shuttling as a major route for activity, cell signaling and behaviorally induced changes in choline uptake and implement a yeast 2-hybrid screen for novel CHT interactors. Finally, we analyze the consequences of full and partial genetic CHT ablation in the mouse for cholinergic biochemistry, pharmacology, physiology and behavior. These studies will elucidate novel aspects of CHT regulation, clarify how CHT supports cholinergic synaptic/behavioral plasticity and provide new CHT links to brain disease.